[Guidelines for medical prescription of medical laboratory tests]. / Recommandations concernant la prescription d'examens de biologie médicale.

The implementation of a computer-assisted prescription is interesting for the laboratory to achieve requirements of NF EN ISO 15189 standard. The test redundancies are also studied and guidelines, founded on validated studies, are proposed. Some solutions concerning the management of orally-formulated prescriptions are given. Finally, a model of collaboration contract between the medical laboratory and the clinical unit is proposed.

[Guidelines concerning stat laboratory process]. / Recommandations concernant le processus des examens urgents.

After a definition of the various emergency situations, vital or organizational, the paper describes the process of urgent laboratory tests requests from the medical prescription until the return of the interpreted results to the clinician. The intervention options of the various professionals to optimize and assure the control of the process 24 hours a day and 7 days/7 are presented. Then, a list of validated available stat tests is proposed. It recovers the main disciplines which have a direct link to bring the help to the clinical teams in responsibilities of critical situations. These propositions must be adapted to the conditions of laboratory local environment.

Seroprevalence of human herpes virus 8 antibody in populations at high or low risk of transfusion, graft, or sexual transmission of viruses.

BACKGROUND: The routes of transmission of human herpes virus 8 (HHV-8) remain unclear. In particular, HHV-8 transmission by blood components and organ transplantation is still debated and raises public health issues. The objective of this study was to determine the prevalence of anti-HHV-8 in selected populations of persons or patients with or without risk factors for the transmission of viral infections, in order to determine the routes of HHV-8 transmission. STUDY DESIGN AND METHODS: A total of 1431 persons or patients at low or high risk of sexually, blood-, or graft-transmitted viral infections were tested by means of a standardized immunofluorescence serologic assay detecting anti-HHV-8. RESULTS: The persons or patients could be classified into three distinct groups according to anti-HHV-8 prevalence: a low prevalence group (0.0% to 5.0%), including healthy blood donors, healthy pregnant women, multiply transfused patients with thalassemia major, and IV drug users; an intermediate prevalence group (5.0% to 20.0%), including organ donors, kidney transplant recipients, and multiply transfused patients with sickle cell disease; a high prevalence group (>20.0%), including HIV-negative persons at high risk of sexually-transmitted viral infections, and HIV-infected homosexual men and heterosexuals. CONCLUSION: The sexual route appears to be the main route of HHV-8 transmission; bloodborne transmission of HHV-8, if it exists, is rare. In contrast, organ transplantation recipients might be exposed to HHV-8 transmission by the transplanted organ, which raises the issue of systematic screening of organ donors.

Association of primary pleural effusion lymphoma of T-cell origin and human herpesvirus 8 in a human immunodeficiency virus-seronegative man.

We describe a case of an 87-year-old human immunodeficiency virus (HIV)-negative man who developed a primary pleural lymphoma without any identifiable tumor mass associated with human herpesvirus 8 (HHV-8) infection. A large T-cell lymphoma was diagnosed based on morphologic, immunophenotypic, and molecular findings. The HHV-8 DNA sequences were detected using specific polymerase chain reaction amplification in the lymphomatous effusion. Study of the patient's serum confirmed the HHV-8 infection. This case report displays the characteristic features of HHV-8-related body cavity-based lymphoma/primary effusion lymphoma previously reported in HIV-seronegative patients, except that it is of T-cell origin. Whether this case may be included or not within the primary effusion lymphoma entity, the association of a pleural T-cell non-Hodgkin lymphoma with HHV-8 infection raises the question of the possible occurrence of T cells as the target of malignant transformation associated with HHV-8 infection.

Bone marrow involvement in lymphomas with hemophagocytic syndrome at presentation: a clinicopathologic study of 11 patients in a Western institution.

Hemophagocytic syndrome (HPS) is a clinicopathologic syndrome that can reveal a non-Hodgkin's lymphoma. The pathologic features of lymphoma associated with HPS remain ill defined. We studied 11 lymphomas associated with HPS on initial bone marrow biopsies, consecutively diagnosed during a 6-year period in a Western institution. There were seven diffuse large B-cell lymphomas (DLBCLs), three T-cell lymphomas (one peripheral T-cell lymphoma unspecified, two hepatosplenic gammadelta T-cell lymphomas [HS gammadeltaTLs]), and one aggressive NK-cell lymphoma/leukemia (NKL). These lymphomas shared common clinicopathologic features with a systemic presentation, a poor outcome (nine patients died within 2 years), and a mild interstitial lymphoid infiltrate of the bone marrow at presentation in nine patients. This equivocal lymphoma infiltrate was blending with normal hematopoietic cells, and CD20 and CD3 immunolabelings were essential for its detection. A high number of reactive T (CD3+) cells, most often with a predominant cytotoxic (CD8+ TiA1+) phenotype, was present in all DLBCLs. By in situ hybridization, Epstein-Barr virus was detected in neoplastic cells of three cases (one DLBCL, one HS gammadeltaTL, and one NKL), which also showed serum viral DNA. Polymerase chain reaction studies disclosed HHV6 DNA sequences in tumor tissues of two DLBCLs, whereas HHV8 DNA was not detected. Because tumor mass indicative of lymphoma was not striking in most patients, bone marrow biopsy appears to be of great value for the diagnosis of an HPS-associated lymphoma, which may be, in Western patients, of B- as well as T- or NK-cell type. Immunostaining for CD3 and CD20 is essential to identify the common subtle lymphoma involvement. Together with a better understanding of the pathogenic processes, an early diagnosis may improve the prognosis of HPS-associated lymphoma.

Busulfan disposition and hepatic veno-occlusive disease in children undergoing bone marrow transplantation.

Hepatic veno-occlusive disease (HVOD) is a frequent life-threatening toxicity in patients undergoing bone marrow transplantation (BMT) after the administration of a high-dose busulfan-containing regimen. Recent studies have shown that the morbidity and mortality of HVOD may be reduced in adults by pharmacologically guided dose adjustment of busulfan. We analyzed the pharmacodynamic relationship between busulfan disposition and HVOD in 61 children (median age, 5.9 years) with malignant disease. Busulfan, given at a dose ranging from 16 mg/kg to 600 mg/m2, was combined with one or two other alkylating agents (cyclophosphamide, melphalan, thiotepa). Only 3 patients received the standard busulfan/cyclophosphamide (BUCY) regimen. A total of 24 patients (40%) developed HVOD, which resolved in all cases. A pharmacokinetics study confirmed the previously reported wide interpatient variability in busulfan disposition but did not reveal any significant alteration in children with HVOD. The mean area under the concentration-time curve (AUC) after the first dose of busulfan was higher in patients with HVOD (6,811 +/- 2,943 ng h ml-1) than in patients without HVOD (5,760 +/- 1,891 ng h ml-1., P = 0.10). This difference reflects the higher dose of busulfan given to patients with HVOD. No toxic level could be defined and, moreover, none of the toxic levels identified in adults were relevant. The high incidence of HVOD in children given 600 mg/m2 busulfan may be linked to the use of more intensive than usual high-dose chemotherapy regimens and/or drug interactions. Before the prospective evaluation of busulfan dose adjustment in children, further studies are required to demonstrate firmly the existence of a pharmacodynamic relationship in terms of toxicity and allogeneic engraftment, especially when busulfan is combined with cyclophosphamide. The maximal tolerated and minimal effective AUCs in children undergoing BMT are likely to depend mainly upon the disease, the nature of the combined high-dose regimen, and the type of bone marrow transplant.

Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies.

Cytochromes P450 (CYP) constitute a superfamily of enzymes involved in the metabolism of xenobiotics. Within the same subfamily, the isoforms present strong similarities, making them difficult to characterize and differentiate. Using heterologous expression in bacteria, five pure human CYP (1A1, 1A2, 2C9, 2E1, 3A4) were easily obtained and used as antigens to raise specific antibodies. These antibodies were characterized for their specificity and sensitivity by immunoblots; anti-CYP3A4 was immunoinhibitor. These antibodies could be used in association with other means to identify the CYPs responsible for production of a given metabolite. The use of our human recombinant CYP1A2 as antigen and the corresponding specific antibody enabled us to quantify the CYP1A2 content in 43 human livers. The average level was 69 pmol of CYP1A2/mg of microsomal proteins. Finally, these antibodies were also used to evaluate the level of heme incorporation in human microsomal CYP expressed in yeasts.

Specificity of in vitro covalent binding of tienilic acid metabolites to human liver microsomes in relationship to the type of hepatotoxicity: comparison with two directly hepatotoxic drugs.

In order to better understand the first steps leading to drug-induced immunoallergic hepatitis, we studied the target of anti-LKM2 autoantibodies appearing in tienilic acid-induced hepatitis, and the target of tienilic acid-reactive metabolites. It was identified as cytochrome P450 2C9, (P450 2C9): indeed, anti-LKM2 specifically recognized P450 2C9, but none of the other P450s tested (including other 2C subfamily members, 2C8 and 2C18). Tienilic acid-reactive metabolite(s) specifically bound to P450 2C9, and experiments with yeast expressing active isolated P450s showed that P450 2C9 was responsible for tienilic acid-reactive metabolite(s) production. Results of qualitative and quantitative covalent binding of tienilic acid metabolite(s) to human liver microsomes were then compared to those obtained with two drugs leading to direct toxic hepatitis, namely, acetaminophen and chloroform. Kinetic constants (Km and Vmax) were measured, and the covalent binding profile of the metabolites to human liver microsomal proteins was studied. Tienilic acid had both the lowest Km and the highest covalent binding rate at pharmacological doses. For acetaminophen and chloroform, several microsomal proteins were covalently bound, while covalent binding was highly specific for tienilic acid and dihydralazine, another drug leading to immunoallergic hepatitis. Although low numbers of drugs were tested, these results led us to think that there may exist a relationship between the specificity of covalent binding and the type of hepatotoxicity.

Busulfan disposition below the age of three: alteration in children with lysosomal storage disease.

Busulfan disposition is age-dependent with a higher clearance and a larger volume of distribution in children than in adults. The optimal dosage of busulfan needed to achieve bone marrow (BM) displacement in young children with malignant or nonmalignant disease remains to be defined. Using a gas chromatography-mass spectrometry assay, we evaluated plasma pharmacokinetics of busulfan in 33 children (median age, 9 months; range, 2 months to 2.75 years) with immune deficiencies, lysosomal storage diseases, acute leukemias, and malignant lymphohistiocytosis after an oral dose ranging from 0.9 to 2.6 mg/kg. The busulfan clearance (assuming a bioavailability of 1) ranged from 2.1 to 13.4 mL/min/kg with a mean of 6.8 mL/min/kg, which is higher than that reported in older children (4.5 mL/min/kg) and adults (2.9 mL/min/kg). Six children with lysosomal storage disease (5 with Hurler's disease, 1 with San Filippo's disease) had a prolonged elimination half-life (4.9 v 2.4 hours), a larger volume of distribution (3.4 v 1.2 L/kg) and a faster clearance (8.7 v 6.3 mL/min/kg) than the other 27 children. This suggests that a higher dose of busulfan will be required to achieve BM displacement in children with lysosomal storage disease. Over the dose range of 0.9 to 2.6 mg/kg, busulfan pharmacokinetics were linear. However, only 46% of the interpatient variation in systemic exposure could be ascribed to the dose. Given the wide interpatient variability in busulfan disposition, dose adjustment and drug monitoring will be needed to achieve the optimal dosage of busulfan in young children. The plasma busulfan levels required to achieve BM displacement need to be defined, especially in lysosomal storage diseases.

Chronopharmacology of high-dose busulfan in children.

In bone marrow transplantation, high-dose busulfan is given p.o., usually every 6 h over 4 consecutive days. Since this repeated administration might alter busulfan disposition, fluctuations in busulfan plasma levels were studied over the 4-day treatment period in 21 children (median age, 5 years) with malignant solid tumors. In addition, urinary excretion of unchanged busulfan was measured every 6 h in 4 patients. Busulfan (37.5 mg/m2 for 16 doses) was given on an empty stomach at 12 p.m., 6 p.m., midnight, and 6 a.m. for 4 consecutive days, starting at 12 p.m. Trough plasma levels, i.e., concentration 6 h after each dose and just before the next one, and urinary excretion of busulfan were measured using a gas chromatography-mass spectrometry assay. Busulfan trough plasma levels exhibited a significant circadian rhythm with a higher mean level at 6 a.m. compared to that at 12 p.m., 6 p.m., and midnight. This rhythm was characterized by a double amplitude (mean +/- SD) of 42 +/- 14% and an acrophase (maximum) occurring at 5:48 a.m. +/- 115 min. In addition, once the steady state was reached, no decreasing trend was observed in any patient. Busulfan renal clearance proved to be low since only 5.4 +/- 1.2% of the given dose were excreted unchanged in urine. In the 4 patients studied, busulfan urinary excretion exhibited a significant circadian rhythm which was apparently linked to the physiological circadian rhythm in urinary output. Ten of 20 evaluable patients developed hepatic venoocclusive disease (HVOD). A significant circadian rhythm in the plasma level was found in both HVOD and non-HVOD patients with no difference between the two groups with regard to the 24-h mean, amplitude, or acrophase. Thus, the circadian changes in busulfan trough plasma levels observed at the steady state were not related to the occurrence of HVOD in these children with solid tumors. Moreover, since this rhythm was stable from day 2 to day 4, it should not compromise dose adjustment.

Is 600 mg/m2 the appropriate dosage of busulfan in children undergoing bone marrow transplantation?

Recent studies have reported that the pharmacokinetics of high-dose busulfan in bone marrow transplantation (BMT) are age-dependent: with the usual dosage of 16 mg/kg over 4 days, systemic exposure is two to four times lower in children than in adults. Data suggested that the dose of busulfan should rather be calculated on the basis of the body surface area (BSA). We measured plasma pharmacokinetics of busulfan in 27 children (mean age, 5.4 years) who were administered a new dosage of 600 mg/m2 over 4 days, ie, 17.8 to 29.2 mg/kg (mean, 24.8 mg/kg), using a gas chromatography-mass spectrometry assay. Our results demonstrate that, with this new dosage, systemic exposure is significantly increased in children compared with that achieved with the usual dosage of 16 mg/kg (6,404 +/- 2,378 v 3,918 +/- 1,170 ng.h/mL; P = .003). Moreover, there is no longer a significant difference in systemic exposure between children treated with this new dosage and adults given a dose of 16 mg/kg of busulfan. However, despite the use of a dosage normalized to the BSA, there is still a wide interindividual variation in systemic exposure, ranging from 3,566 to 13,129 ng.h/mL, which may account for the high incidence of venoocclusive disease (VOD) of the liver that we have already reported. The optimal dosage and schedule of busulfan in children requires a more individual approach that could be based on dose adjustment and plasma level monitoring.

Dose-dependent neurotoxicity of high-dose busulfan in children: a clinical and pharmacological study.

Busulfan is known to be neurotoxic in animals and humans, but its acute neurotoxicity remains poorly characterized in children. We report here a retrospective study of 123 children (median age, 6.5 years) receiving high-dose busulfan in combined chemotherapy before bone marrow transplantation for malignant solid tumors, brain tumors excluded. Busulfan was given p.o., every 6 hours for 16 doses over 4 days. Two total doses were consecutively used: 16 mg/kg, then 600 mg/m2. The dose calculation on the basis of body surface area results in higher doses in young children than in older patients (16 to 28 mg/kg). Ninety-six patients were not given anticonvulsive prophylaxis; 7 (7.5%) developed seizures during the 4 days of the busulfan course or within 24 h after the last dosing. When the total busulfan dose was taken into account, there was a significant difference in terms of neurotoxicity incidence among patients under 16 mg/kg (1 of 57, 1.7%) and patients under 600 mg/m2 (6 of 39, 15.4%) (P less than 0.02). Twenty-seven patients were given a 600-mg/m2 busulfan total dose with continuous i.v. infusion of clonazepam; none had any neurological symptoms. Busulfan levels were measured by a gas chromatographic-mass spectrometry assay in the plasma and cerebrospinal fluid of 9 children without central nervous system disease under 600 mg/m2 busulfan with clonazepam:busulfan cerebrospinal fluid:plasma ratio was 1.39. This was significantly different (P less than 0.02) from the cerebrospinal fluid:plasma ratio previously defined in children receiving a 16-mg/kg total dose of busulfan. This study shows that busulfan neurotoxicity is dose-dependent in children and efficiently prevented by clonazepam. A busulfan dose calculated on the basis of body surface area, resulting in higher doses in young children, was followed by increased neurotoxicity, close to neurotoxicity incidence observed in adults. Since plasma pharmacokinetic studies showed a faster busulfan clearance in children than in adults, this new dose may approximate more closely the adult systemic exposure obtained after the usual 16-mg/kg total dose, with potential inferences in terms of anticancer or myeloablative effects. The busulfan dose in children and infants undergoing bone marrow transplantation should be reconsidered on the basis of pharmacokinetic studies.